Functional Proteomics for biomarker characterisation

Discovery of biomarkers has advanced significantly in the past few years due to the new developments in Genomics, Proteomics, Metabolomics and other systematic approaches. However, one area of systematic biological analysis that is still relatively underserved is the rapid elucidation of the biological functions of a proteome.

Let’s take a step back. Proteins are present in the cell, but they also have a function. Therefore, in some instances, the mere identification of the presence of a protein may not be enough to elucidate the biological pathways involved in a given disease. It is for this that activity assays are needed.

Activity assays are also an area where major technological developments have been done. However, we are still missing a high-throughput approach allowing to measure the enzymatic activity of several hundred proteins at the same time. What we could call an “activity profile” of a given sample, allowing us to compare it with a control sample. In two words, Functional Proteomics.

From “Detection” Proteomics to “Functional” Proteomics

Classical Proteomic approaches include 2D electrophoresis followed by MassSpec (MS), especially for unknown proteins (for known proteins, we already have antibody arrays, which are faster and more convenient).

A variety of techniques has been developed to allow study of enzymatic activities following MS (1, 2). In general, they rely on the identification of phosphorylated peptide substrates by MS, using either purified enzymes (e.g. kinases) or cell extracts. This means that the substrates must be known, so these techniques may lack profiling power when substrates are not know. Alternative approaches include a more global profiling of kinase activities (3) without prior knowledge of the substrates, but they still only characterise kinases. Their use has not been validated for other enzymatic activities.

In order to overcome these limitations, a new approach has been developed by Wang and co-workers. Basically, by performing 2D electrophoresis in non-denaturing conditions, they guarantee that the 3D structure (and the activity) of all proteins in a biological sample is retained. A new protein-recovery method was developed to collect the separated proteins from the 2D gel into microplates to facilitate systematic protein function analysis and MS identification. This new approach is called PEP technology.

PEP technology for functional proteomcis. Proteins are separated by IEF and subjected to a modified 2D gel process. The large-format PEP plate has 1,536 wells. Following PEP transfer and collection, proteins from each well are analyzed by standard SDS-PAGE or MS.

PEP for Functional Proteomics

In brief, using the PEP technology from Array Bridge, proteins are first separated by Iso-Electric Focusing (IEF), followed by a modified 2D gel process. No reducing agent is used, and a refolding process is implemented after the IEF step. Then, proteins are eluted into the “PEP plate”, using a recovery solution to reduce protein diffusion from the plate as well as to help protect protein function. PEP plates come in different formats to allow fitting even large-format 2D gels.

The format of the plates is aimed at getting high-resolution, so that each well contains one or just a few proteins.

This is new by itself. But what comes after is very interesting too!

Following PEP transfer and collection, the proteins from each well can be analysed for protein function and also for ulterior studies (we will see that later).

As far as enzymatic activities are concerned, this technology has been developed for several type of enzymes (NADH oxidases, NADPH oxidases, Kinases and Proteinases, either in large or small format), allowing to have a complete profiling of the activity landscape in a given sample.

This allows Life scientists to have a Functional Proteomics profile of samples of interest, and compare them with a control sample (e.g. study a cancer sample vs. a healthy one, or see the effect that your drug has in the enzymatic pathways in the cell…).

Furthermore, only a part of the PEP-eluted sample can be used for ulterior MS studies to exactly identify the exact proteins being present in each well, and responsible for the activity seen before.

If more than one protein is present in the well, one can also use standard SDS-PAGE for size determination.

PEP technology can be used for the systematic analysis of metabolic enzymes. In this example, NADH-dependent oxidase is analyzed from a proteome of interest.

Examples of studies where this technology has been used include the obtaining of a snapshot of NADH oxidase activities, activity-based biomarker discovery, developing of new protein kinase inhibitors, or just a simple protein purification tool.